This invention generally relates to methods of treating cancer, and particularly to methods of treating cancer using dithiocarbamate derivatives.
Cancer, the uncontrolled growth of malignant cells, is a major health problem of the modem medical era and ranks second only to heart disease as a cause of death in the United States. While some malignancies, such as adenocarcinoma of the breast and lymphomas such as Hodgkin""s Disease, respond relatively well to current chemotherapeutic antineoplastic drug regimens, other cancers are poorly responsive to chemotherapy, especially non-small cell lung cancer and pancreatic, prostate and colon cancers. Even small cell cancer of the lung, initially chemotherapy sensitive, tends to return after remission, with widespread metastatic spread leading to death of the patient. Thus, better treatment approaches are needed for this illness. Also, because almost all currently available antineoplastic agents have significant toxicities, such as bone marrow suppression, renal dysfunction, stomatitis, enteritis and hair loss.
The end of the twentieth century has seen a more dramatic increase in the observed incidence of malignant melanoma than for all other types of tumors. The biology of malignant melanomas offers an example of the importance of transcription factors for malignant cell propagation. Malignant melanomas have great propensity to metastasize and are notoriously resistant to conventional cancer treatments such as chemotherapy and xcex3-irradiation. Development of malignant melanoma in humans progresses through a multistage process, with transition from melanocyte to nevi, to radial growth, and subsequently to the vertical growth, metastatic phenotype of autonomous melanomas, associated with decreased dependence on growth factors, diminished anchorage dependence, reduced contact inhibition and increased radiation and drug resistance.
Much of the molecular understanding of melanoma progression has come from studying the response of cultured melanoma cells to mitogenic stimuli. In culture, melanocyte proliferation and differentiation are positively regulated by agents that increase cAMP (See, P. M. Cox, et al., xe2x80x9cAn ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II Drxcex1 promoter and activation by SV40 T-antigen,xe2x80x9d Nucleic Acids Res. 20:4881-4887 (1992); R. Halaban, et al., xe2x80x9cRegulation of tyrosinase in human melanocytes grown in culture,xe2x80x9d J. Cell Biol. 97:480-488 (1983); D. Jean, et al., xe2x80x9cCREB and its associated proteins act as survival factors for human melanoma cells,xe2x80x9d J. Biol. Chem. 273:24884-24890 (1998); P. Klatt, et al., xe2x80x9cNitric oxide inhibits c-Jun DNA binding by specifically targeted S-glutathionylation,xe2x80x9d J. Biol. Chem. 274:15857-15864 (1999); J. M. Lehmann, et. al., xe2x80x9cMUC18, a marker of tumor progression in human melanoma, shows sequence similarity to the neural cell adhesion molecules of the immunoglobulin superfamily,xe2x80x9d Proc. Natl. Acad. Sci. U.S.A. 89:9891-9895 (1989); M. Luca, et al., xe2x80x9cDirect correlation between MUC18 expression and metastatic potential of human melanoma cells,xe2x80x9d Melanoma Res. 3:35-41(1993); J. P. Richards, et al., xe2x80x9cAnalysis of the structural properties of cAMP-responsive element-binding protein (CREB) and phosphorylated CREB,xe2x80x9d J. Biol. Chem. 271:13716-13723 (1996); and S. Xie, et al., xe2x80x9cDominant-negative CREB inhibits tumor growth and metastasis of human melanoma cells,xe2x80x9d Oncogene 15:2069-2075 (1997)), and several cAMP responsive transcription factors binding to CRE (the consensus motif 5xe2x80x2-TGACGTCA-3xe2x80x2, or cAMP response element) play prominent roles in mediating melanoma growth and metastasis. In MeWo melanoma cells, the transcription factor CREB (for CRE-binding protein) and its associated family member ATF-1 promote tumor growth, metastases and survival through CRE-dependent gene expression. See, D. Jean, et al., supra. Expression of the dominant negative KCREB construct in metastatic MeWo melanoma cells decreases their tumorigenicity and metastatic potential in nude mice. See, S. Xie, et al., xe2x80x9cExpression of MCA/MUC18 by human melanoma cells leads to increased tumor growth and metastasis,xe2x80x9d Cancer Res. 57:2295-2303 (1997). The KCREB-transfected cells display a significant decrease in matrix metalloproteinase 2 (MPP2, the 72 kDa collagenase type IV) mRNA and activity, resulting in decreased invasiveness through the basement membrane, an important component of metastatic potential.
The cell surface adhesion molecule MCAM/MUC18, which is involved in metastasis of melanoma (See, J. M. Lehmann, et al., supra; M. Luca, et al., supra; S. Xie, et al., supra), is also down-regulated by KCREB transfection. See, S. Xie, et al., Cancer Res., supra. In addition, expression of KCREB in MeWo cells renders them susceptible to thapsigargin-induced apoptosis, suggesting that CREB and its associated proteins act as survival factors for human melanoma cells, thereby contributing to the acquisition of the malignant phenotype. See, D. Jean, et al., supra.
Melanoma cells aberrantly express the major histocompatibility complex class II (MHC II) antigens, normally found only in B-lymphocytes and antigen presenting cells of the monocyte/macrophage cell line. See, P. M. Cox, et al., xe2x80x9cAn ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II Drxcex1 promoter and activation by SV40 T-antigen. Nucleic Acids Res.,xe2x80x9d 20:4881-4887 (1992). In B16 melanoma cells this is due to activation of the MHC II DRxcex1 promoter by constitutive activation of an ATF/CREB motif. CREB family proteins also bind to the UV-response element (URE, 5xe2x80x2-TGACAACA-3xe2x80x2), and URE binding of the CREB family member ATF2 confers resistance to irradiation and to the chemotherapeutic drugs cis-platinum, 1xcex2-D-arabinofuranosylcytosine (araC) or mitomycin C in MeWo melanoma lines. See, Z. Ronai, et al., xe2x80x9cATF2 confers radiation resistance to human melanoma cells,xe2x80x9d Oncogene 16:523-531 (1998)). Thus, CREB family transcription factors play important roles in the malignant potential of this important tumor type. This has led to the suggestion by others that targeted molecular disruption of ATF/CREB-mediated transcription might be therapeutically useful for controlling growth and metastases of relatively treatment-resistant malignant melanoma. See, D. Jean, supra, and Z. Ronai, supra.
The positively charged DNA binding domain of many transcription factors contains cysteines which can be oxidatively modified by agents such as hydrogen peroxide or nitric oxide (NO), stimulating repair processes that result in formation of mixed disulfides between glutathione (GSH) and protein thiols. See, P. Klatt, et al., supra; and H. Sies, xe2x80x9cGlutathione and its role in cellular functions,xe2x80x9d Free Rad. Biol. Med. 27:916-921 (1999)). As a consequence of this so-called protein xe2x80x9cS-glutathionylationxe2x80x9d, the usually positively charged transcription factor DNA binding domain develops an electronegative charge imparted by dual carboxylate end groups of GSH. The change in charge disrupts transcription factor binding to its respective DNA consensus sequence. See, P. Klatt, et al., supra and H. Sies, supra. This mechanism has been demonstrated to explain how NO inhibits c-Jun DNA binding by specifically targeted S-glutathionylation of cysteines within the DNA binding region, and a similar mechanism has been suggested for how nitrosative stress in general might functionally inhibit the activity of Fos, ATF/CREB, Myb and Rel/NFxcexaB family transcription factors. See, P. Klatt, et al., supra.
The dithiocarbamates comprise a broad class of molecules giving them the ability to complex metals and react with sulfhydryl groups and glutathione. After metal-catalyzed conversion to their corresponding disulfides, dithiocarbamates inhibit cysteine proteases by forming mixed disulfides with critical protein thiols. See, C. S. I. Nobel, et al., xe2x80x9cMechanism of dithiocarbamate inhibition of apoptosis: thiol oxidation by dithiocarbamate disulfides directly inhibits processing of the caspase-3 proenzyme,xe2x80x9d Chem. Res. Toxicol. 10:636-643 (1997). CREB contains three cysteines in the DNA binding region (Cys300, Cys310 and Cys337) which are not essential for DNA binding but might provide reactive sites for S-glutathionylation. See, S. Orrenius, et al., xe2x80x9cDithiocarbamates and the redox regulation of cell death,xe2x80x9d Biochem. Soc. Trans. 24:1032-1038 (1996)).
Recently, dithiocarbamates containing a reduced sulfhydryl group, e.g., pyrrolidinedithiocarbamate (PDTC) have been shown to inhibit the proliferation of cultured colorectal cancer cells. See, Chinery, et al., xe2x80x9cAntioxidants enhance the cytotoxicity of chemotherapeutic agents in colorectal cancer: a p53-independent induction of p21WAF1/CIP1 via C/EBPxcex2,xe2x80x9d Nature Med. 3:1233-1241 (1997); Chinery et al., xe2x80x9cAntioxidants reduce cyclooxygenase-2 expression, prostaglandin production, and proliferation in colorectal cancer cells.xe2x80x9d Cancer Res. 58:2323-2327 (1998).
In addition to their reduced thioacid form, dithiocarbamates exist in three other forms, e.g., a) the disulfide, a condensed dimmer of the thioacid, with elimination of reduced sulfhydryl groups by disulfide bond formation; b) the negatively charged thiolate anion, generally as the alkali metal salt, such as sodium; and c) the 1,1-dithiolato complexes of the transition elements, in which the two adjoining sulfur atoms of the dithiocarbamate are bound to the same titanium, vanadium, chromium, iron, cobalt, nickel, copper, silver or gold metal ion. The disulfide, thiolate anion and transition metal complexes of dithiocarbamates are all structurally distinct from the reduced form of PDTC used by Chinery, et al., in that they have no reduced sulfhydryl molecular moiety and are incapable of functioning as antioxidants by donating the proton from a reduced sulfhydryl to scavenge electrons of free radical species. Lacking a reduced sulfhydryl, thiocarbamate disulfides, thiolate anions and transition metal complexes should, according to the teachings of Chinery, et al., have no activity as antiproliferative compounds against cancer, since these three nonreduced chemical forms of dithiocarbamates are incapable of functioning as antioxidants.
In U.S. patent application Ser. No. 09/392,122; filed Sep. 8, 1999, it was reported that the dithiocarbamate disulfide disulfiram sensitizes tumor cells to cancer chemotherapy and could be used in conjunction with cancer chemotherapeutic drugs to increase their effectiveness in treating neoplasms. Recently, this effect has been explained in work in which disulfiram was shown to prevent maturation of the P-glycoprotein pump, an ATP-driven 170-kd efflux pump on the plasma membrane that pumps a variety of cytotoxic drugs out of cells. See, T. W. Loo, et al., xe2x80x9cBlockage of drug resistance in vitro by disulfiram, a drug used to treat alcoholism.xe2x80x9d J. Natl. Cancer Inst. 92:898-902 (2000). This effect reduces P-glycoprotein-mediated drug resistance in tumor cells and sensitizes tumor cells to cancer chemotherapy.
It is therefore an object of the present invention to provide a method for the treatment of cancer.
Another object of the present invention is to provide pharmaceutical compositions for the treatment of cancer.
It is still another object of the present invention to provide a relatively less toxic agent available for use alone in combination with current drugs in order to better treat cancer patients without risking injury from the therapy itself.
The present invention provides a method for treating established cancer using dithiocarbamate disulfides, or thiocarbamate anions either alone, or in combination with a heavy metal ion, and thiocarbamate complexes of heavy metal ions.
It has been discovered that dithiocarbamate disulfides and their corresponding thiolate anions alone exhibit potent inhibitory effects on growth of established tumor cells in the absence of antioxidant sulfhydryl groups within their structure. Thiocarbamate disulfides and their corresponding thiolate anions are effective in inhibiting the growth of established melanomas and non-small cell lung cancer cells, which are known to be poorly responsive to currently available neoplastic agents. In addition, it has further been surprisingly discovered that the antiproliferative and antineoplastic effect of dithiocarbamate disulfides and their corresponding thiolate anions on established tumor cells is greatly potentiated by co-treatment of cancer cells with a transitional metal salt in a concentration which by itself does not impair cancer cell growth. The potentiating function of the transition metal is to facilitate formation of the thiolate anion from the dithiocarbamate disulfide. Further the tumor cell growth inhibition effect can be significantly enhanced by the addition of heavy metal ions such as copper, zinc, gold and silver ion, as examples, or by administering the thiocarbamate as a heavy metal ion complex.
The chemical activity of these species is not from antioxidant action but from stimulating formation of mixed disulfides between the dithiocarbamate and sulfhydryl moieties of cysteines located at critical sites on cell proteins, such as the DNA binding region of transcription factors needed to promote expression of gene products necessary for malignant cell proliferation.
Dithiocarbamates disulfides that are useful in the treatment of cancer include, but are not limited to, those of the formulas:
R2R3N(S)CSxe2x80x94SC(S)NR2R3 
wherein R1, R2, R3 and R4 are the same or different and represent hydrogen, and unsubstituted or substituted alkyl, akenyl, aryl, alkoxy, and heteroaryl groups. It is noted that the alkyl groups can include cycloalky and hetercycloalkyl groups. R1, R2 and the N atom in the formula can together form an N-heterocyclic ring, which is, e.g., heterocycloalkyl or heterocycloaryl. Likewise, R3, R4 and the N atom in the formula can together form an N-heterocyclic ring, which is, e.g., heterocycloalkyl or heterocycloaryl. Typically R1 and R2 are not both hydrogen, and R3 and R4 are not both hydrogen.
In accordance with another aspect of this invention, a method for treating established cancer in a patient is provided comprising administering to the patient a therapeutically effective amount of a dithiocarbamate disulfide, preferably disulfiram, or the corresponding diethyldithiocarbamate thiolate metal complex of the formula: 
wherein R2 and R3 are the same or different and represent hydrogen, and unsubstituted or substituted alkyl, akenyl, aryl, alkoxy, and heteroaryl groups; M is a heavy metal, e.g., arsenic, bismuth, gallium, manganese, selenium, zinc, titanium, vanadium, chromium, iron, cobalt, nickel, copper, silver or gold; n is the valence of the metal, and An is an anion selected from the group consisting of chloride, bromide, iodide, acetate and low molecular weight organic or inorganic pharmaceutically acceptable anions.
In a preferred embodiment, the heavy metal ion is administered as a complex or chelate with the dithiocarbamate disulfide or corresponding thiolate anion. Suitable heavy metal ions include but are not limited to ions of arsenic, bismuth, cobalt, copper, chromium, gallium, gold, iron, manganese, nickel, silver, titanium, vanadium, selenium, and zinc.
In another preferred embodiment, the dithiocarbamate disulfide or corresponding thiolate anion and the heavy metal ion are administered in combination with another anticancer agent.
In addition, the present invention provides a method for sensitizing cancer cells to chemotherapeutic drugs by the administration of a dithiocarbamate thiolate anion or a dithiocarbamate complex with heavy metals in order to effect inhibition of the tumor cell membrane P-glycoprotein pump which functions to extrude from cancer cells the anti-neoplastic agents which are absorbed.
In accordance with another aspect of the invention, the present invention provides a pharmaceutical composition that comprises a pharmaceutically acceptable carrier, and a complex between a dithiocarbamate and a heavy metal ion. Optionally, the composition can further contain another anticancer agent.
The active compounds of this invention can be administered through a variety of administration routes. For example, they can be administered orally, intravenously, intradermally, subcutaneously and topically.
The present invention is effective for treating various types of cancer, including but not limited to melanoma, non-small cell lung cancer, small cell lung cancer, renal cancer, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, ovarian cancer, uterine cancer, lymphoma, and prostate cancer. In particular the present invention will be especially effective in treating melanoma, lung cancer, breast cancer and prostate cancer. Thus the use of dithiocarbamate disulfides and thiolate anions in this invention offers a readily available and easily used treatment for cancers in man and other animals.